Particle detection system and components thereof

ABSTRACT

A combined impedance and fluorescence particle detection system including an optically transmissive plate having an orifice for the flow of particles therethrough, a light source which operably directs light on a particle at the orifice, and a light detector positioned so as to detect light which is emitted by the particle, and wherein the plate acts as a waveguide to direct light along part of its path between the light source and light detector.

The invention relates to a particle detection system in particular sucha system comprising an impedance particle detector and fluorescencedetector.

It is known to detect particles such as blood cells or yeast cells forexample within a sample by passing the particles through a narroworifice and detecting variations in the impedance across the orifice.Additionally, it is known to dye or stain samples with a suitablefluorescent dye and then illuminate the particles with a suitable sourceof light such as laser light of a fundamental frequency and thereafterdetermine the nature of the particles by the fluorescence signal emittedfrom the particles.

However, such known systems are very complex, costly, require continuousadjustment and are limited in terms of the minimum size of particlesthat can be detected. Accordingly, the invention seeks to improveimpedance and fluorescent particle detection systems preferably makingthem more economical to manufacture and/or more efficient to operate. Anobject is to simplify the optical system and integrate the optics withthe impedance system, especially by using an orifice plate as part ofthe fluorescence and impedance systems.

According to one aspect of the invention there is provided a combinedimpedance and fluorescence particle detection system comprising anoptically transmissive plate having an orifice for the flow of particlestherethrough, a light source which operably directs light on a particleat the orifice, and a light detector positioned so as to detect lightwhich is emitted by the particle, and wherein the plate acts as awaveguide to direct light along part of its path between the lightsource and light detector. Beneficially therefore the plate comprises anorifice to effect the impedance measurement which plate also acts as awaveguide for part of the optical system. For example light can betransmitted from the orifice to a detector via the plate and/or to theorifice via the plate from a light source.

The direction of flow of particles passes through the plate, however, atleast part of the light path between the source and detector can be indifferent direction with respect to the particle flow direction. In oneparticular form, light is projected from the light source substantiallyin line with the particle flow direction and the light detector issubstantially at right angles thereto. In another form, the source anddetector are substantially in line, therefore on opposite sides of theorifice plate.

Preferably the system is operable at two or more fundamental lightfrequencies to observe fluorescence. The light source can comprise atleast one individual unit which emits light at different frequencies.Also or alternatively, more than one light source is provided enablinguse of two different wavelengths of light. Beneficially this enablesdifferent properties of the particles to be measured. Preferably adetector is provided for each light source in order to determine thefluorescence at a given wavelength.

In a preferred form, a light source and/or a light detector is opticallycoupled to the orifice plate. The light source and/or detector can bedirectly optically coupled to the orifice plate. However, preferably thelight detector can be directly optically coupled to a filter which isdirectly optically coupled to the orifice plate. Preferably the platecomprises a substantially straight edge for attachment of or coupling ofthe light sources and/or light detectors. In preferred forms, theorifice plate is polygonal especially of quadrilateral, hexagonal oroctagonal shape. None, one or more, indeed all, edges of the orificeplate can carry a light source or detector. The plate can also be discshaped.

To optimise efficiency of light transfer to the detector the waveguideproperties of the plate are preferably optimised. The plate surfacessuch as faces and/or edges can be treated so as to increase internalreflections within the plate. For example, the faces and/or edges can becoated such as with silver or aluminium.

In a preferred form, at least part of the plate edge is so treated so asto increase reflections towards the detector. In a preferred form bothfaces of the plate are partially treated so as increase internalreflections.

Accordingly, fluorescent light initially scattered away from thedetector can be reflected by the silvered edge back towards thedetector.

Preferably, the orifice is located in a region of relatively highconcentration of internally reflected light, possibly a focal point ofthe plate. For example, for a disc-shaped plate this can be a centralposition or where coated surfaces are used, an off-centre position.Combinations of the following three features are possible (to providesix possibilities): the orifice is positioned at a point of increasedconcentration of internal reflections within the plate, one or moreedges of the plate are treated especially by coating to increaseinternal reflection, one or more faces of the plate are treated such asby metallic coating to increase internal reflection.

The plate for example can be a ruby, quartz or sapphire crystal, orother optically transparent medium. Preferably the refractive index ofthe plate is higher than saline or other media such as diluent used tocarry or dilute the sample particles. Preferably, the surface finish onthe plate is smooth to a quarter wavelength.

Preferably a filter is positioned between the plate and the detector inorder to attenuate frequencies other than the fluorescence emissionfrequency from the particles. Accordingly, the filter is preferably aband pass filter wherein the optimum transmission is based on theemission frequency from the particles which is of course shifted awayfrom the fundamental frequency of the light source and or thecharacteristics are chosen to maximise the difference in attenuationbetween the emissive frequency from the particles and the fundamentalfrequency of the light source.

Preferably, the optically transmissive orifice plate is made of onepiece but can be made of components or parts such as a first orificecarrying part mounted in a larger mount or slide part to improvehandling and positioning of the plate within the particle detectionsystem. Such a mounting part can for example be a glass slide andpreferably the first part is optically bonded to the mount part using asuitable adhesive having a refractive index similar to that of theorifice carrying part of the plate. Preferably the surfaces and/or edgesof the orifice carrying part and/or the mount part are treated so as toincrease internal reflections, and optimise transfer of light to thedetector or from the light source.

Another aspect of the invention provides a light waveguide or anoptically transmissive plate for a particle detection system which platecomprises an orifice for allowing flow of particles through the plateand part of the extremities or surfaces of the plate are treated so asto increase internal optical reflections within the plate. The platecomprises an optically transparent region adjacent or surrounding theorifice thereby to allow input and output of light to and from theplate. Additionally, at least part of the extremities of the plate arealso optically transparent to enable attachment of a light source ordetector to said part.

Embodiments of the invention will now be described, by way of exampleonly, with reference to the accompanying drawings, in which:

FIG. 1 is a schematic block diagram of a system according to theinvention;

FIG. 2 is a schematic perspective view of the orifice plate assembly andpart of the optical system according to the invention;

FIGS. 3 and 4 are schematic front and side elevations of an orificeplate according to the invention in a slightly different form to thatshown in FIG. 2; and

FIG. 5 is a schematic perspective view of another embodiment of theinvention.

Referring to FIG. 1 it can be seen that a particle detection system 10according to the invention comprises a controller 12 having amicroprocessor 14 which operably communicates with an impedancedetection circuit 16, laser controller 18, and light detector circuit20. The controller 12 further comprises a power input line P andsuitable means for powering each of sub-systems 14 to 20, and at leastone input/output port I/O for example for communication with a displayor printer.

System 10 further comprises a sample chamber 22 having a pair ofelectrodes 24, one disposed in each of compartments 26 and 28. Thecompartments 26 and 28 are separated by a plate 30 comprising an orifice44 (see FIG. 2; the orifice 44 is not shown in FIG. 1 for clarity),which allows the flow of particles between the compartments 26 and 28. Alight source such as a laser 31 is provided for directing a light beam Lat the orifice 30. The light source 31 is controlled by laser controller18 and preferably emits a coherent light beam L principally of afundamental frequency or wavelength. For example laser 31 can be a lowcost semiconductor laser. The laser 31 can have a fundamental wavelengthof between say 300 and 700 nm.

System 10 further comprises a light detector 32 in optical contact withplate 30 and separated therefrom by a light filter 34. Detector 32operably communicates with detection circuit 20.

Referring to FIGS. 2 to 4, greater detail of part of the plate 30 isshown. Plate 30 is made up of a mount 36 such as a glass slide. Theouter faces and edges of the glass slide 36 are preferably treated toincrease internal reflections, such as by silvering. However, mount 36comprises an untreated edge 38 which abuts filter 34 which is in opticalcommunication with detector 32.

Mount 36 further comprises an aperture 40 for receiving a disc 42 havingan orifice 44. The disc 42 is preferably optically coupled to an edge ofmount 36 which defines aperture 40 for example using a suitablerefractive adhesive. Accordingly plate 30 can comprise a first orificecarrying part such as disc 42 optically mounted in a second mount part36.

Disc 42 is shown in greater detail in FIGS. 3 and 4 where it can be seenthat in its preferred form disc 42 is a fairly flat disc shape, forexample, having a thickness T of about 30 to 200 microns more preferably75 to 180 microns and more preferably still 80 to 160 microns, and adiameter D in the order of 5 to 15 millimeters and more preferably 10millimeters. Disc 42 can comprise a ruby or sapphire crystal forexample.

Disc 42 further comprises front and back faces 46 and an outer edge 48.Preferably, the front and back faces are treated so as to increaseinternal reflections within the disc 42. An aperture or region Ysurrounding orifice 44 can be left untreated. For example, the diameterof aperture Y can be in the order of a millimeter while the diameter oforifice 44 might be in the order of 30 to 80 microns. Preferably theedge 48 is treated between the positions X marked on the circumferenceof disc 42 so as to increase internal reflections adjacent orifice 44thereby directing light towards light detector 32, i.e. reflecting backto the right of the page as viewed in FIG. 3.

In this example, shown in FIG. 3, where half the edge 48 of plate 42 issilvered, orifice 44 is located halfway between the centre of thecircular disc and the silvered edge 48. But the orifice 44 can becentrally located as shown in FIG. 2.

In use, a sample is placed in one of compartments 26 or 28 and particlesare drawn through orifice 44 into the other compartment. The size of theaperture 44 is such as to allow individual particles to flow betweencompartments 26 and 28. This movement of particles through orifice 44 isdetected due to variation of impedance between electrodes 24 i.e.through the electrolyte within compartments 26 and 28. This is detectedusing circuit 16 and microprocessor 14 to count the number of particlesfor example and/or size the particles flowing between the compartments.Additionally, microprocessor 14 and circuit 18 are used to drive lightsource 31 so as to direct a light beam L at the orifice 44. By suitablydyeing or staining the particles in a sample using a fluorescent dye,the particles passing through orifice 44 emit light at a differentfrequency to the fundamental frequency of the light source 31. At leastpart of the emitted fluorescent signal is captured by disc 42surrounding orifice 44. Disc 42 thereafter acts as a waveguide to directthe emitted light towards light detector 32. This waveguide action isassisted by the silvered faces 46 of disc 44 and the silvered edge 48.

The light transmitted by disc 42 is transferred into mount 36 and inturn to edge 38 thereafter into filter 34 and eventually on to detector32. Beneficially since mount 36 is made of an optically transmissivematerial such as glass, and has silvered faces and edges, the amount oflight emitted by particles at orifice 44 which reaches detector 32 isoptimised.

A further embodiment of a suitable light source, light detector andorifice plate arrangement according to the invention is shown in FIG. 5.In this embodiment like features with the earlier embodiments are giventhe same two digit reference number as earlier prefixed with thedigit 1. Accordingly, optically transmissive plate 130 comprises anaperture 144. Plate 130 is an integral plate made from opticallytransmissive material such as a ruby or sapphire crystal. Plate 130 inthis example is rectangular wherein the edges of the plate haveoptically contacted therewith, for example by using an opticallytransmissive adhesive, an optical component of the fluorescence system.Here, a light source 131 a such as a laser is attached to the upper edgeof plate 142 and a suitable light detector 132 a is attached via filter134 a to a side edge of plate 130. An associated light source and lightdetector operate respectively to project light on to particles passingthrough orifice 144 and detect emitted fluorescent light scatteredthrough plate 130 via filter 134 a to detector 132 a Similarly, a secondlight source 131 b is attached to the lower edge of plate 130 and anassociated detector and filter 132 b and 134 b are attached to the otherside edge of plate 142 to enable detection of fluorescent light ofsuitable wavelengths emitted from the particles passing through orifice144. The front and rear faces of plate 130 can of course be treated toincrease internal reflections.

Plate 130 of this example can be of a two or more part constructionalso. The waveguide properties of the plate are a result of the plateactive co-operation in directing light between the light source andlight detector due for example to total internal reflections at thesurface of the plate which reflections can be enhanced by coatings.Beneficially also, the solid angle at the orifice surface defined by theedge of the plate at the orifice is optimised to allow sufficientcapture of emitted light from a particle. Preferable, the surfacedefined in the orifice is continuous and therefore formed integrally inpart of the plate.

In another form of the invention, edge 38 is directly coupled to abundle of fibreoptic cables. That part of edge 38 not in optical contactwith the end of an optical fibre is masked so as to optimise capture oflight inpinging on the edge. In addition, preferably an interferencefilter is positioned between the optical fibres and the detector toselect the fluorescent signal over any background signal for example.Beneficially, the fibreoptic cables act to collimate the light andenable transmission of the fluorescent signal to a detector somewhatremote from mount 36.

What is claimed is:
 1. A combined impedance and fluorescence particledetection system comprising an optically transmissive plate having anorifice for the flow of particles therethrough, a light source whichoperably directs light on a particle at the orifice, and a lightdetector positioned so as to detect light which is emitted by theparticle, and wherein the plate acts as a waveguide to direct lightemitted by the particle along at least part of its path between theorifice and the light detector.
 2. A system according to claim 1 whereinthe light source operably projects light substantially in line with theparticle flow direction and the light detector is out of line therewithand preferably substantially at right angles thereto.
 3. A systemaccording to claim 2 wherein the light is projected against thedirection of flow of particles in use.
 4. A system according to claim 3wherein the plate is adapted substantially to block transmission oflight from the light source to the sample other than through theorifice.
 5. A system according to claim 1 wherein light is transmittedto the orifice via the plate from a light source.
 6. A system accordingto claim 5 wherein the light source and light detector are substantiallyin line, on opposite sides of the orifice plate.
 7. A system accordingto claim 1 operable at two or more different wavelengths of light.
 8. Asystem according to claim 7 comprising at least one light emitting unitwhich is operable at two ore more different fundamental frequencies. 9.A system according to claim 7 comprising two or more light sources. 10.A system according to claim 7 wherein a detector is provided for eachlight source and/or fundamental incident frequency in order to determinethe fluorescence at a given wavelength.
 11. A system according to claim1 wherein at least one of a light source and a light detector isoptically coupled to the orifice plate.
 12. A system according to claim11 wherein a light source and/or detector is directly optically coupledto the orifice plate.
 13. A system according to claim 1 wherein thelight detector is directly optically coupled to a filter which isdirectly optically coupled to the orifice plate.
 14. A system accordingto claim 1 wherein the orifice plate comprises a substantially straightedge for at least one of the light sources and the light detectors. 15.A system according to claim 1 wherein the orifice plate is polygonallyshaped.
 16. A system according to claim 15 wherein one or more edges ofthe orifice plate carries at least one of a light source and a lightdetector.
 17. A system according to claim 1 wherein the waveguideproperties of the orifice plate are enhanced by treating part of theorifice plate surfaces to increase internal reflections.
 18. A systemaccording to claim 17 wherein the treated surface comprises a reflectivecoating such as a metallic coat.
 19. A system according to claim 17wherein at least part of the orifice plate edge is so treated so as toincrease reflections.
 20. A system according to claim 17 wherein thefaces of the plate are partially treated so as to increase internalreflections.
 21. A system according to claim 1 wherein the orifice islocated in a region of the plate of relatively high concentration ofinternally reflected light.
 22. A system according to claim 1 having anorifice plate wherein at least one of the orifices is positioned at apoint of increased concentration of internal reflections within theplate, one or more edges of the plate are treated especially by coatingto increase internal reflection, and one or more faces of the plate aretreated such as by metallic coating to increase internal reflection. 23.A system according to claim 1 wherein the orifice plate has a refractiveindex higher than saline or any other media such a diluent used to carryor dilute the sample particles which media operably surrounds theorifice.
 24. A system according to claim 1 wherein a filter ispositioned between the plate and the detector in order to attenuatefrequencies other than the fluorescence emission frequency from theparticles.
 25. A system according to claim 24 wherein the filter is aband pass filter wherein the characteristics are chosen to maximise thedifference of attenuation between the emissive frequency from theparticles and the fundamental frequency of the light source.
 26. Asystem according to claim 1 wherein the optically transmissive orificeplate is an integral one piece construction.
 27. A system according toclaim 1 wherein the orifice plate comprises a first orifice carryingpart mounted in a second mount part.
 28. A system according to claim 27wherein the first part is optically bonded to the second part using asuitable adhesive having a refractive index similar to that of the firstpart of the plate.
 29. A system according to claim 1 comprising anoptical fibre between the plate and detector.
 30. An opticallytransmissive plate for a particle detection system which plate comprisesan orifice for allowing flow of particles through the plate and part ofthe extremities or surfaces of the plate are treated so as to increaseinternal optical reflections within the plate.
 31. An opticallytransmissive plate for a particle detection system, the plate comprisingan orifice for allowing a flow of particles through the plate andwherein part of the extremities or surfaces of the plate are treated soas to increase internal optical reflections within the plate and toenable the plate to act as a waveguide to direct light emitted by theflow of particles along at least part of its path between the orificeand an external surface of the plate.
 32. A combined impedance andfluorescence particle detection system comprising a plate having anorifice for the flow of particles therethrough and comprising anoptically transmissive material, a light source which operably directslight on a particle at the orifice, and a light detector positioned soas to detect light which is emitted by the particle, orifice, and alight detector positioned so as to detect light which is emitted by theparticle, and wherein the plate acts as a waveguide to direct lightemitted by the flow of particles along at least part of its path betweenthe orifice and light detector through the optically transmissivematerial.